1. Field of the Invention
The invention is generally related to pharmaceuticals, which are useful for allosterically modifying pyruvate kinase.
2. Description of the Prior Art
Mammalian pyruvate kinase (PK) is a key regulatory glycolytic enzyme that exhibits allosteric kinetic behavior. The basic mechanism of the allosteric regulation of PK at the molecular level is still not known. There are reports that PK undergoes conformational changes and that the changes involve domain events. PK is found in all cells and tissues. It catalyzes the conversion of phospho-enolpyruvate (PEP) and adenosine diphosphate (ADP) into pyruvate and adenosine triphosphate (ATP), as shown in reaction scheme 1.
PEP+Mg.ADP+H+xe2x86x92(Mg2+, K+)xe2x86x92Mg.ATP+Pyruvatexe2x80x83xe2x80x83(1)
The reaction proceeds in two steps. First, the 2-phosphate is removed from PEP to yield ATP and the enolate ion form of pyruvate. The second step involves the protonation and tautomerization of the ion to yield the keto form of pyruvate. The enzyme requires three cation cofactors, two divalent (Mg2+ or Mn2+) and one monovalent (K+). Positive factors of PK include fructose 1,6 diphosphate, PEP, and low pH. Negative factors of PK include ATP, high pH, and glycogenic amino acids such as alanine and phenylalanine. The products of reaction scheme 1, pyruvate and ATP, are involved in a wide variety of metabolic pathways; therefore, PK can be considered a key enzyme in the glycolytic pathway as well as many other pathways in the cellular metabolism.
In mammals, PK has four isoenzymes, which are identified as M-1, M-2, L and R type. The R type PK exists exclusively in red blood cells and its biochemical properties change with cell maturation. Congenital R-PK deficiency in erythrocytes is one of the most frequent enzymopathies involving the glycolytic pathway. It is an autosomal recessive disorder, and in hemozygotes, causes nonspherocytic hemolytic anemia. Heterozygotes do not show symptoms of hemolytic anemia, but have lowered residual PK activity, with increased 2,3-diphosphoglycerate (2,3-DPG) and fairly stable ATP levels. Thus, it appears quite possible that similar results will be obtained pharmacologically from partial inhibition of erythrocyte PK. Because ATP can be synthesized in erythrocytes only by glycolysis, a decreased PK activity causes severe disturbances of the erythrocyte energy metabolism and leads to greatly diminished lifetime of the red blood cells.
It is an object of this invention to provide pharmaceuticals suitable for allosterically modifying PK.
It is another object of this invention to use a family of pharmaceuticals to regulate the 2,3-DPG and ATP levels in vivo.
It is yet another object of this invention to use a family of pharmaceuticals to regulate the glycolytic pathway in vivo.
According to the invention, a family of compounds has been identified which allosterically modify pyruvate kinase. A compound within the family will be useful for the delivery of additional oxygen to tissues by increasing the 2,3-DPG concentration in vivo, and this can be useful in a wide variety of clinical conditions and disease states including radiation oncology, whole body and tissue hypothermia, hypoxia, chronic hypoxia, sepsis, wound healing, diabetic ulcers, pressure sores, tissue transplants, stroke, shock, cardiovascular ischemia and angina applications, acute respiratory distress syndrome (ARDS), chronic respiratory insufficiency, pulmonary fibrosis, interstitial lung disease, peripheral vascular disease (e.g., intermittent claudication), ischemia, including Alzheimer""s disease (AD). PK contributes to the establishment of steady-state levels of 2,3-DPG, which is important since 2,3-DPG is an allosteric effector of oxygen binding to hemoglobin. PK was found to have an inverse relationship with 2,3-DPG levels in human erythrocytes. An increase in the level of 2,3-DPG induces a rightward shift of the oxygen-hemoglobin dissociation curve, indicating that the quaternary conformational equilibrium of hemoglobin is perturbed toward the T, or deoxygenated state. In other words, oxygen is more quickly being delivered from blood to tissues. Similarly, a decrease in the level of 2,3-DPG concentration induces a leftward shift of the oxygen-hemoglobin dissociation curve and shifts the allosteric equilibrium to the R, or oxygenated state. Such agents will be useful as antisickling agents. Increasing erythrocyte 2,3-DPG concentrations through the intervention of PK inhibition will find use in many clinical settings where more delivery of tissue oxygenation is desired.
A chemical modification of hemoglobin in vivo may provide an exciting alternative in the treatment of stroke in humans by enhancing oxygen delivery and reducing penumbral injury. Our past studies with allosteric inhibitors have produced exciting clinical results in treating acute hypoxia. However, chronic treatment of hypoxia with hemoglobin allosteric effectors is limited due to the high daily doses required to treat the large amount of Hb in red cells (5 mM in red cells). An alternate method to treat chronic hypoxia would be to inhibit the breakdown of the natural allosteric effector of Hb, 2,3-DPG. The much lower preponderance of red cell pyruvate kinase to Hb levels makes this an interesting target for treating chronic hypoxia. Besides the obvious chronic hypoxic diseases, treatment of AD is practically unknown as a focus for hypoxic therapy. However, Regelson and Harkins have pinpointed studies that firmly indicate that an increase in oxygen to the brain of AD patients may well improve the degree of severity of the disease. Regelson, W., Harkins, S. W., xe2x80x9cAmyloid is not a tombstonexe2x80x9dxe2x80x94a summation. The primary role for cerebrovascular and CSF dynamics as factors in Alzheimer""s disease (AD): DMSO, fluorocarbon oxygen carriers, thyroid hormonal, and other suggested therapeutic measures, Ann. NY Acad. Sci. 826: 348-374 (1997). Pappas et al. have shown that chronic low-grade ischemia in rats can damage learning and memoryxe2x80x94resembling clinical AD. Pappas, B. A., Davidson, C. M., Bennett, S. A., de la Torre, J. C., Fortin, T., Tenniswood, M. P., Chronic ischemia: memory impairment and neural pathology in the rat, Ann. NY Acad. Sci. 826:498-501 (1997). Olichney et al. have found clinical correlations between cerebral hemorrhage, infarction, and later amyloid vascular deposits, suggesting that amyloid in the vessel wall can contribute to ischemic pathology. Olichney, J. M., Ellis, R. J., Katzman, R., Sabbagh, M. N., Hansen, L., Types of cerbrovascular lesions associated with severe cerebral amyloid angiopathy in Alzheimer""s disease, Ann. NY Acad. Sci. 826: 493-497 (1997). Cohen et al. support this work in showing that AD vascular pathology relates to angiopathy with amyloid adventitiously extending from the small vessels as an intrinsic part of the blood vessel wall. Cohen, D. L., Hedera, P., Premkumar, D. R., Friedland, R. P., Kalaria, R. N., Amyloid-beta protein angiopathies masquerading as Alzheimer""s disease? Ann. NY Acad. Sci. 826:390-395 (1997). Other work also suggests that defects in cerebral vascular blood flow are directly and indirectly associated with AD. Moroney, J. T., Bagiella, E., Hachinski, V. C., Molsa, P. K., Gustafson, L., Brun, A., Fischer, P., Erkinjuntti, T., Rosen, W., Paik, M. C., Tatemichi, T. K., Desmond, D. W., Misclassification of dementia subtype using the Hachinski ischemic score: results of a meta-analysis of patients with pathologically verified dementias, Ann. NYAcad. Sci. 826:490-492 (1997); Hoyer, S., Models of Alzheimer""s disease: cellular and molecular aspects, J. Neural Transm. Suppl. 49:11-21 (1997). Decreased blood flow (and thus decreased oxygen delivery) is directly related to the severity of AD. To date, it has not been clinically possible to increase oxygen delivery to brain, except for the acute use of an allosteric effector of hemoglobin. Teicher, B. A., Wong, J. S., Takeuchi, H., Gravelin, L. M., Ara, G., Buxton, D., Allosteric effectors of hemoglobin as modulators of chemotherapy and radiation therapy in vitro and in vivo, Cancer Chemother. Pharmocol, 42:24-30 (1998). Therefore, these compounds could also be of use in partial or even a higher degree of success in treatment of brain disorders such as AD and stroke. Also, the blood brain barrier would not restrict the efficacy of allosteric PK inhibitors as the effectors do not have to cross the blood brain barrier, but will act directly on PK in the red cell to increase oxygen delivery.